Abstract:

Disclosed is a method for total synthesis of CTX1B, which is developed for
the synthesis of a ciguatoxin analogue such as CTX3C and enables the more
efficient application of an established reaction to the total synthesis
of CTC1B. More specifically, disclosed is a method for total synthesis of
CTX1B comprising; an 0.5-acetal formation for synthesizing a novel
compound (3); a radical cyclization reaction for constructing a
9-membered ring formation reaction including a novel compound (6) through
a novel compound (8) and yielding a compound (D); and a deprotection for
yielding CTX1B. Also disclosed are novel compounds (1) to (8) which are
particularly useful for synthesis of CTX1B and can be used for the
synthesis of a ciguatoxin analogue.

Claims:

1. A method for preparation of CTX1B including following 10 processes,
##STR00028## a double bond of compound A ##STR00029## is oxidized by
osmium tetra oxide and changed to diol derivative of compound A,
transformed the diol to aldehyde by oxidation cleavage by sodium
periodate, then reduced the aldehyde to alcohol using sodium borohydride
and obtain compound 1 (process 1), ##STR00030## alcohol of compound 1 is
transformed to compound 2 using diphenyldisulfide•tributylphosphine
(process 2), ##STR00031## said compound 2 is transformed to
α-chrolosulphide and synthesize compound B, ##STR00032## under the
presence of DTBMP said compound B is joined to the ABCDE ring segments
compound C as O,S-acetal using silver triflate and compound 3 is formed
(process 3), ##STR00033## TIPS group of said compound 4 is removed using
TBAS and form compound 4 (process 4), ##STR00034##
pentafluorophenylpropiolate is joined to alcohol of said compound 4 using
trimethylphosphine and compound 5 is formed (process 5), ##STR00035##
carry out radical cyclizing reaction on said compound 5 by treating with
AIBN and tintributyl hydride and form G ring part, so that compound 5
transforms to carboxylic acid compound D, then ##STR00036## transforms to
methyl ester by acting trimethylsilyldiazomethane and forms compound 6
(process 6), ##STR00037## forms compound 7 (process 7) by reducing methyl
ester of said compound 6 by diisobutylaluminum hydrate under lower
temperature condition, then transforms to olefin by Wittig reaction,
##STR00038## forms compound 8 (process 8) by forming F ring part by
carrying out ring closure methathesis reaction acting Grubbs catalyst to
said compound 7, ##STR00039## synthesizes compound E, 1,2-diol of A ring
side chain of which is protected by naphthylacetal, by oxidizing 6 NAP
groups using DDQ and removing 5 NAP groups (process 9), then ##STR00040##
carries out acid treatment on said compound E (process 10),wherein, in
compounds A, B, D, E, CTX1B and compounds 1-8, NAP is 2-naphthylmethyl
group, Me is methyl group, TIPS is triisopropylsilyl group, Ph is phenyl
group, further, shortened mark DTBMP is 2,6-di-butyl-4-methylpyridine,
TBAF is tetrabutylammonium fluoride, AIBN is
α,α'-azobis(isobutyronitrile), DDQ is
2,3-dichloro-5,6-dicyano-1,4-benzoquinone.

2. A novel compound represented by compound 1 to be used for preparation
of CTX1B of claim 1.

3. A novel compound represented by compound 2 to be used for preparation
of CTX1B of claim 1.

4. A novel compound represented by compound 3 to be used for preparation
of CTX1B of claim 1.

5. A novel compound represented by compound 4 to be used for preparation
of CTX1B of claim 1.

6. A novel compound represented by compound 5 to be used for preparation
of CTX1B of claim 1.

7. A novel compound represented by compound 6 to be used for preparation
of CTX1B of claim 1.

8. A novel compound represented by compound 7 to be used for preparation
of CTX1B of claim 1.

9. A novel compound represented by compound 8 to be used for preparation
of CTX1B of claim 1.

Description:

FIELD OF THE INVENTION

[0001]The present invention relates to the establishment of a method for
total synthesis of ciguatoxin CTX1B which is a homolog of ciguatoxin,
further, relates to a provision of compounds useful to make possible an
effective method for preparation of said total synthesis.

BACKGROUND OF THE INVENTION

[0002]Food-poisoning, ciguatera caused by poisoning of originally
non-toxic fishes, widely occurs in coral reef islands region of
subtropical and tropical regions, and more than 50,000 people suffer
annually from ciguatera. Although the mortality is not so high, symptoms
such as abnormal sensation, diarrhea, lassitude, arthralgia or itching
last for several months under some circumstances. Ciguatoxins (CTX),
which are isolated and the structure of which is decided as a main
originated poison of ciguatera, are macromolecules characterized by fused
13 ether rings and their molecular length is approximately 3 nm, further
more than 20 kinds of homolog are existing. Ciguatoxins are produced from
dinoflagellate Gambierdiscus toxicus and accumulate in fishes by means of
food chain. Since approximately 400 kinds of toxic fishes are normal from
the view points of appearance, taste and odor, it is not safe to exploit
fish sources of southern sea region. Therefore, the development of
detective method of ciguatoxins by means of easy and high sensitive
immunological measuring method of ciguatoxins is strongly expected.

[0003]Ciguatoxins bind specifically to voltage-sensitive Na.sup.+ channels
(VSSC) of excitable membranes, activate it and generate toxicity,
however, the activation mechanism of ciguatoxins at structural level is
not made clear yet. Ciguatoxins exist in nature is very small and
cultural production by the dinoflagellate is very slow, detail biological
research and the preparation of anti-CTX antibody using natural product
is virtually impossible. Under said circumstances, the quantitative
supply of natural ciguatoxins by practical chemical synthesis is strongly
desired.

[0004]Inventors of the present invention already proposed a total
synthesis of CTX3C, which is one of main homolog of ciguatoxin (non
patent document 1, Proc. Natl. Acad. Sci. U.S.A. 101, 1203-12018 (2004)).
Further, the inventors developed a Sandwich immunoassay that can detect
CTX3C easily (non patent document 2, J. Am. Chem. Soc. 125, 7608-7612
(2003)) and are now investigating to apply it to identification of a
ciguatera fish. However, since CTX3C is mainly contained in a herbivorous
fish, preparation of an antibody originated to other homolog is necessary
for detection of ciguatoxin from a carnivorous fish.

[0005]CTX1B is the most typical ciguatoxin contained mainly in a
carnivorous fish and has more complicated structure than CTX3C, and is
known as the most historically important ciguatoxin whose structure is
firstly decided in 1989. At the decision of structure, 0.3 mg of CTX3B
isolated from 4000 kg of poisonous moray is used. However, since it was
actually impossible to obtain practical amount of sample from nature,
development of total synthesis of CTX1B is awaited for the actual use of
CTX1B as a standard sample.

[0006]Generally, in total synthesis, if partial structure is different,
development of a new synthesis route becomes necessary. However, for the
purpose to synthesis many ciguatoxin homologs existing in nature in a
unified fashion, the inventors have developed convergent total synthesis,
which is characterized to be remarkably simple and more reliable compared
with competitive methods. By said method, supply of over than several mg
of CTX3C became possible up to this time. According to said concept,
since there is possibility that carnivorous fishes accumulate ciguatoxin
by higher concentration than herbivorous fishes because carnivorous
fishes are locating at upper position of food chain than herbivorous
fishes and is more dangerous as a ciguatera poisoned fish, the inventors
of the present invention considered to develop a new effective total
synthesis of CTX1B for the purpose of investigation of CTX1B.

[0007]At the development of a new effective total synthesis, the inventors
of the present invention considered to utilize the reaction sequence
which were already developed for synthesis of CTX3C. Namely, the
inventors considered to apply the coupling of ABCDE ring segments with
HIJKLM ring segments and subsequent construction of FG ring to CTX1B.
However, since 7-members ring E-structure, and a side chain existing in A
ring segments of CTX1B are structurally different from CTX3C, direct
application of methodology used in CTX3C was impossible. Therefore, the
inventors planned to develop a higher yielding process from a view point
of effective preparation of the aimed compound.

[0008](1) At the formation of 7-members ring of compound D at radical ring
forming reaction, preparation process of CTX3C can not be used.
Therefore, the inventors designed compound 5 that has pentafluoroacrylate
instead of conventionally used methylacrylate and yield of ring forming
reaction is remarkably improved.

[0009](2) At deprotection of naphthylmethyl (NAP) group, side chain of A
ring segment is very unstable to acid and compound E acetal intermediate
is formed at conventional acid hydrolysis of acetal. Therefore, various
investigations for condition are carried out and it is understood that
acetal can be removed by condition of 1N hydrochloric acid/methanol, and
the total synthesis of CTX1B can be carried out for the first time.
Further, at above mentioned development, the inventors considered to
utilize HIJKLM ring segments compound A, which was already reported in a
paper (non-patent document 3, J. Org. Chem. 69, 2797-2804 (2004)), and
compound C (non-patent document 4, J. Org. Lett., 6, 751-754 (2004)) as
one of intermediates, according to the thinking that O,S-acetal compound
3, which is the most important intermediate, can be synthesized by
coupling reaction developed by the inventors that permits neutral
condition.

SUBJECT OF THE INVENTION

[0010]The subject of the present invention is to provide an effective
method for total synthesis of CTX1B by high yield. Aiming to accomplish
said subject, the inventors of the present invention considered that the
designing of an intermediate that can apply an established reaction to be
considered rational in synthesis of ciguatoxin analogous compound is
important. That is, the subject of this invention is to provide an useful
compound that can be used for an effective method for total synthesis of
CTX1B, further to link to an improvement of synthesis of ciguatoxin
analogous compound. From said points of view, the inventors of the
present invention continued investigation and adopted O,S-acetal forming
reaction that synthesizes compound 3. and 9-rings forming reaction from
compound 6 to 8, further, developed a radical ring-forming reaction to
obtain afore mentioned compound D and a deprotection reaction to obtain
CTX1B newly, and by synthesizing all new compounds to link the
intermediate to aimed compound, and can accomplish afore mentioned
subject.

DISCLOSURE OF THE INVENTION

[0011]The first one of the present invention is a method for synthesizing
the aimed compound of CTX1B including following 10 processes. First
process is comprised of oxidizing double bond in compound A by using
osmium tetra oxide to change to a diol derivative of compound A, and
after transforming the diol to an aldehyde by oxidation cleavage by using
sodium periodate, reducing the aldehyde to alcohol using sodium
borohydride to obtain compound 1 (process 1).

##STR00001##

Second process is transforming the alcohol of compound 1 to compound 2
using diphenyldisulfide•tributylphosphine (process 2).

##STR00002##

Third process comprises of transforming the compound 2 to
α-chrolosulphide to synthesize the compound B, and synthesizing
compound 3 by joining the ABCDE ring segments compound C (Refer to the
non-patent document 4, J. Org. Lett., 6, 751-754 (2004)) and the compound
B as O,S-acetal using silver triflate (AgOTf) (process 3), under the
presence of DTBMP (dimethylsulfooxide).

##STR00003##

(process 3).

##STR00004##

Forth process is forming the compound 4 by removing TIPS
(triisopropylsilyl) group from the compound 3 using TBAF
(tetrabutylammonium fluoride) (process 4).

Sixth process comprises of transforming the compound 5 to carboxylic acid
compound D by forming G ring part by carrying out radical cyclizing
reaction on said compound 5 treating by AIBN
(α,α'-azobis(isobutyronitrile)) and tributyltin hydride, and

##STR00007##

transforming to methyl ester by acting trimethylsilyldiazomethane and to
form compound 6 (process 6).

Ninth process is synthesizing compound E, 1,2-diol of A ring side chain of
which is protected by naphthylacetal, by oxidizing 6 NAP
(2-naphthylmethyl) groups using DDQ
(2,3-dichloro-5,6-dicyano-1,4-benzoquinone) and removing 5 NAP groups
(process 9).

[0012]The second one of the present invention is a novel compound
represented by compound 1, which is useful for the method to prepare
CTX1B. The third one of the present invention is a novel compound
represented by compound 2, which is useful for the method to prepare
CTX1B. The fourth one of the present invention is a novel compound
represented by compound 3, which is useful for the method to prepare
CTX1B. The fifth one of the present invention is a novel compound
represented by compound 4, which is useful for the method to prepare
CTX1B. The sixth one of the present invention is a novel compound
represented by compound 5, which is useful for the method to prepare
CTX1B. The seventh one of the present invention is a novel compound
represented by compound 6, which is useful for the method to prepare
CTX1B. The eighth one of the present invention is a novel compound
represented by compound 7, which is useful for the method to prepare
CTX1B. And the ninth one of the present invention is a novel compound
represented by compound 8, which is useful for the method to prepare
CTX1B.

EFFECT OF THE INVENTION

[0013]The offering of an effective total synthesis of CTX1B of the present
invention is important from the view point that practical amount of said
compound to ensure a progress in the research of biological science or a
development for detection of Ciguatera poisoned fish can be supplied, and
has an effect that can be practically used as a standard specimen of
Ciguatera food-poisoning happened in all over the world.

PREFERRED EMBODIMENT OF THE INVENTION

[0014]The present invention will be illustrated more in detail. A. Since
HIJKLM ring segments compound A (J. Org. Chem. 69, 2797-2804 (2004)),
which was reported in afore mentioned non-patent document 3, has a
structure corresponding to half of CTX1B, said compound A is used as an
intermediate for synthesis of CTX1B. By reaction condition mentioned in
following reaction formula, double bond of compound A is oxidized by
osmium tetra oxide and transformed to diol, then transformed to aldehyde
by oxidation cleavage by sodium periodate (at room temperature), after
that, reduced to alcohol using sodium borohydride and obtain compound 1
(yield of these two processes is 91%).

[0016]Compound 2 is transformed to a α-chrolosulphide in 6:1 mixed
solvent of carbon tetrachloride and dichloromethane using NSC and
compound B is synthesized. Then, ABCDE ring segments compound C, which is
already reported in paper (afore mentioned non-patent document 4, J. Org.
Lett., 6, 751-754 (2004)), and compound B are joined as O,S-acetal using
silver triflate (AgOTf) in 1:5 mixed solvent of carbon tetrachloride and
dichloromethane under the presence of DTBMP and compound 3 is obtained
(yield to compound C is 63%).

##STR00014##

[0017]TIPS group of compound 3 is removed by TBAF and compound 4 is formed
(yield is 92%). Pentafluorophenylpropiolate acrylate is introduced into
alcohol of compound 4 using pentafluorophenylpropiolate and
trimethylphosphine and compound 5 is formed (yield is 94%).

##STR00015##

[0018]Radical cyclizing reaction is carried out on compound 5 by treating
with AIBN and tributyltin hydride in toluene at 85° C. and G ring
part is formed, thus carboxylic acid compound D is obtained. In mixed
solvent of benzene and methanol, trimethylsilyldiazomethane is acted to
compound D so as to transform to methyl ester, and compound 6 is formed.

##STR00016##

[0019]Methyl ester of compound 6 is reduced to aldehyde by
diisobutylaluminum hydride under low temperature condition, then
transformed to olefin by Wittig reaction and compound 7 is formed. Grubbs
catalyst is acted to compound 7 and F ring part is formed by carrying out
ring closure methathesis reaction, and compound 8 is obtained.

##STR00017##

[0020]Six NAP groups of compound 8 are oxidized using DDQ and 5 NAP groups
is removed, thus compound E characterized that 1,2-diol of A ring side
chain is protected by naphthylacetal is formed. Finally, compound E is
treated by 1N hydrochloric acid in methanol solvent and total synthesis
of CTX1B, which is aimed compound, is accomplished.

##STR00018##

EXAMPLES

[0021]More concrete synthesis will be shown as Examples, however, these
Examples are shown to understand the present invention more easily and
not intending the scope of the present invention.

CH2Cl2 (5.0 mL, 0.25M) solution of aldehyde is cooled to
0° C., and sodium borohydride (25 mg, 670 μmol) is added and
stirred for 30 minutes. Reaction is stopped by adding saturated
NH4Cl aqueous solution into this solution and diluted by ethyl
acetate. Water phase is extracted by ethyl acetate for 3 times, and
combined organic layer is washed by saturated brine, then is dried by
Na2SO4. Solvent is concentrated and refined by a flash column,
then alcohol of compound 1 (138 mg, 1.17 μmol) is obtained. Total
yield of this 2 processes is 91%. Features of compound 1 are shown in
Table 1.

[0035]DIBAL solution (0.9M hexane solution, 17 μL, 15.1 μmol) is
dropped slowly to compound 6 (3.2 mg, 1.51 μmol), which is cooled down
to -100° C., stirred for 30 minutes and elevate the temperature to
-90° C. Reaction is stopped by adding Ethyl acetate and saturated
NH4Cl aqueous solution and diluted by ethyl acetate. Water layer is
extracted by ethyl acetate for 3 times and combined organic layer is
washed by saturated brine and dried by Na2SO4. Solvent is
concentrated and crude aldehyde is obtained. This aldehyde is not refined
and used in next reaction.

THF (1.0 mL, 0.001M) of triphenylphosphonium bromide (54 mg, 151 μmol)
is treated with t-BuOK (8.4 mg, 75 μmol) at 0° C., and mixture
is stirred at 0° C. for 20 minutes. THF solution (0.5 mL) of
aldehyde is introduced and is stirred at 0° C. for 30 minutes.
Reaction is stopped by adding saturated NH4Cl aqueous solution and
water solution is extracted by ethyl acetate. Organic layer is washed
with saturated brine, then dried by Na2SO4. Solvent is
concentrated and refined by a flush column and hexaene (2.3 mg, 1.16
μmol, total of two process is 77%) of compound 7 is obtained. Features
of compound 7 are shown in Table 7.

[0039]DDQ (1.6 mg, 6.9 μmol) is added to CH2Cl2 (100
μL)-water (100 μL) solution of compound 8 (450 μg, 0.23 μmol)
and stirred at room temperature for 45 minutes. Na2S2O3
aqueous solution is added and reaction is stopped, and diluted by ethyl
acetate and saturated NaHCO3 aqueous solution. Water phase is
extracted by ethyl acetate for 5 times and combined organic layer is
washed by saturated brine, then solvent is concentrated. Obtained mixture
is refined by HPLC and compound E is obtained. Hydrochloric acid (1N. 50
μL) is added to methanol (200 μL) solution of compound E is added
and stirred at room temperature for 30 minutes. Saturated NaHCO3
aqueous solution is added to this solution and reaction is stopped, then
concentrated. This mixture is diluted by water and ethyl acetate for 5
times, and combined organic layer is concentrated. Crude CTX1B is refined
by HPLC and CTX1B (108 μL mg, 0.097 μmol, 42%) is obtained.
Features of synthesized CTX1B are shown in Table 9.

[0057]The present invention makes possible to provide necessary amount of
said compound for progressing bioscience research or development of
method for detection of ciguatera poisoned fish, and is useful for
industrial use applicable as a standard sample for ciguatera
food-poisoning happened in all over the world.